IL-1 is a pro-inflammatory cytokine which play an important role in the activation and regulation of host defence and immune responses to inflammation or injury. IL-1 is able to bind and activate IL1-RI and IL1-RII, which are found on many cells types. The role of the IL-1 signalling in the deployment of Th cell subsets, especially Th17 cells is well known. However, the specific cells which are responsible for the expression of IL-1 signalling in the immune defense and in the development of the Th cell lineage in response to infection, is still largely unclear. Therefore in this thesis, IL1-RI conditional knockout mice specifically in hematopoietic cells (IL1-RI vaviCre+) were generated. Using IL1-RI vaviCre+ mice in comparison with IL1-RI global knockout mice (IL1-RI-/-) would determine whether the expression IL-1 signalling from hematopoietic cells is responsible for the immune defense and in the development of the Th1, Th2 and Th17 cells against gastrointestinal helminth Trichuris muris (T.muris) infections. The generation of IL1-RI vaviCre+ mice have been investigated at the genomic and proteomic level in order to confirm that the Il1-rI gene is inactivated in hematopoietic cells. The characterisation of IL1-RI vaviCre + mice at the genomic level confirmed that the Il1-rI gene was obliterated successfully. At protein level the characterisation of IL1- RI vaviCre + mice confirmed that IL1-RI was dysfunctional in hematopoietic cells. Additionally, the development of the immune cells was investigated in IL1-RI vaviCre + and IL1-RI-/- mice. Our findings demonstrated that the lymphocyte development was not affected by the deletion of the IL1- RI gene. This data indicated that IL1- RI vaviCre + and IL1-RI-/- mice are vital in vivo models. In high dose infection, both IL1-RI vaviCre + and IL1-RI -/- mice were able to clear the infections due to their ability to generate a Th2 response. Both IL1-RI vaviCre + and IL1-RI -/- mice infected with low dose of T.muris were susceptible to infections and showed high levels of Th1 cytokines. Thus, we hypothesised that IL1-RI signalling in hematopoietic cells was not required for worm expulsion and the generation of Th2 and Th1 response. Interestingly, low dose T.muris infection showed a clear reduction in the Th17 cytokines IL22 and IL17 in both IL1-RI vaviCre + and IL1-RI -/- mice, suggesting that IL-1 signalling expressed from hematopoietic cells is responsible for the development of Th17 cells and secretion of IL17 and IL22. IL1- RI vaviCre + and IL1-RI -/- mice infected with low dose of T.muris also showed an increase in inflammation in the colon and decreased of goblet cell hyperplasia. It is well known that IL22 plays an important role in preventing tissue damage and repair. Thus, in this study IL22 global knockout mice (IL22 -/-) were used to determine if the change in crypt lengths and goblet cell hyperplasia in IL1-RI vaviCre + and IL1-RI -/- was due to an absence of IL22. Our finding showed that IL22 -/- mice infected with low dose of T.muris had increased crypt length and a reduction in goblet cells. The similar phenotype in crypt length and goblet cell hyperplasia between IL22 -/-, IL1-RI vaviCre + and IL1-RI -/- mice suggested that a lack of IL22 in IL1-RI vaviCre + and IL1-RI -/- mice is responsible for the change in mice phenotype. It also provides more evidence for the role of IL-1 signaling in hematopoietic cells in the generation of Th17 cells and in the production of its cytokine IL22.IL1-RII is an inhibitor of IL1-RI, thus, in this study IL1-RII global knockout mice (IL1-RII -/-) mice was used in comparison with IL1-RI -/- mice to verify the role of IL-1 signaling in the development of Th17 cells. Our finding showed an overexpression of IL17 and IL22 in IL1-RII -/- compared with IL1-RI -/- mice and a higher level of IL17 in IL1-RII -/- mice compared with IL1-RII flox/flox mice. This data confirmed that IL-1 signaling is important for the development of Th17 cells and the production of its cytokine IL17 and IL22.

Submitted by MARCELA PETROLINI CAPOBIANCO (mpcapobianco@yahoo.com.br) on 2017-04-24T11:43:11Z No. of bitstreams: 1 TESE MARCELA PETROLINI CAPOBIANCO.pdf: 3177156 bytes, checksum: 6b6af4a4ea15e5048723eabdb0659191 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-04-25T20:25:33Z (GMT) No. of bitstreams: 1 capobianco_mp_dr_sjrp.pdf: 3177156 bytes, checksum: 6b6af4a4ea15e5048723eabdb0659191 (MD5) / Made available in DSpace on 2017-04-25T20:25:33Z (GMT). No. of bitstreams: 1 capobianco_mp_dr_sjrp.pdf: 3177156 bytes, checksum: 6b6af4a4ea15e5048723eabdb0659191 (MD5) Previous issue date: 2017-04-11 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / No Brasil o Plasmodium vivax é a espécie mais prevalente, responsável por aproximadamente 85% dos casos de malária. Ademais as variantes da proteína circumesporozoíta (CSP - VK210, VK247 e P. vivax-like) já foram identificadas em várias áreas endêmicas no país. Diversos estudos analisando a influência da variabilidade genética de receptores celulares e moléculas envolvidas na resposta imune, com diferentes peptídeos do Plasmodium, têm obtido resultados variáveis de acordo com o antígeno utilizado e a população analisada. Este trabalho apresenta resultados sobre o estudo de polimorfismos genéticos no TCR (T cell Receptor) e nas Interleucinas 1 e 2 em pacientes infectados por P. vivax provenientes do município de Goianésia do Pará, no Estado do Pará. Avaliou-se estes polimorfismos com a parasitemia do indivíduo, com os genótipos da CSP e com a resposta sorológica contra os peptídeos da CSP. Foram relacionados também estes polimorfismos com os níveis de citocinas. Os polimorfismos foram analisados por técnicas de PCR-RFLP e PCR alelo específico. As análises sorológicas da CSP foram realizadas por ELISA. Foram comparadas as frequências genotípicas observadas segundo o teorema de Hardy-Weinberg (HW). Os níveis de IL1 e IL2 foram avaliados por citometria de fluxo, seguindo protocolo descrito pelo fabricante. Associação dos polimorfismos com os níveis de interleucinas foi avaliada por Análise de variância. As frequências genotípicas e alélicas foram obtidas no programa R v 2.11.1. A parasitemia variou de 15 a 70.000, com mediana de 1.500 parasitos/mm3. Os SNPS investigados mostraram frequências variadas na amostra estudada. Todo o polimorfismo avaliado encontra-se em Equilíbrio de Hard Wenberg. Não houve diferença significativa da parasitemia em relação aos SNPs investigados. Infecções contendo apenas a variante VK247 foram as mais comuns e também não foi observado diferença significativa na resposta de anticorpos de acordo com a variante da CSP presente no momento da infecção. Correlações significativas entre os níveis destas interleucinas com a parasitemia e os níveis de anticorpos contra as variantes da CSP não foram observadas. Além disso, as variantes da CSP não influenciaram os níveis plasmáticos das citocinas e não houve associações positivas entre os SNPs das IL1 e IL2 e seus níveis plasmáticos. Os resultados poderão contribuir na identificação e participação efetiva de genes humanos na modulação da resposta imune, essenciais no estabelecimento de estratégias de imunização contra a doença, em área de transmissão ativa no Estado do Pará. / In Brazil, Plasmodium vivax is the most prevalent species responsible for approximately 85% of malaria cases. In addition, variants of the circosporozoite protein (CSP - VK210, VK247 and P. vivax - like) have already been identified in several endemic areas in the country. Several studies analyzing the influence of the genetic variability of cellular receptors and molecules involved in the immune response with different Plasmodium peptides have obtained variable results according to the antigen used and the analyzed population. This work presents results on the study of genetic polymorphisms in TCR (T cell Receptor) and in Interleukins 1 and 2 in patients infected by P. vivax from the city of Goianésia do Pará, in the State of Pará. These polymorphisms were evaluated with Parasitemia, CSP genotypes and serological response to CSP peptides. These polymorphisms were also related to cytokine levels. Polymorphisms were analyzed by PCR-RFLP and allele-specific PCR techniques. Serological tests of CSP were performed by ELISA. The genotypic frequencies observed according to the Hardy-Weinberg (HW) theorem were compared. Levels of IL1 and IL2 were evaluated by flow cytometry following the protocol described by the manufacturer. Association of polymorphisms with interleukin levels was evaluated by analysis of variance. The genotypic and allelic frequencies were obtained in program R v 2.11.1. The parasitemia ranged from 15 to 70,000, with a median of 1,500 parasites / mm3. The SNPS investigated showed varied frequencies in the sample studied. All polymorphism evaluated is in Hard Wenberg Equilibrium. There was no significant difference in parasitemia in relation to the investigated SNPs. Infections containing only the VK247 variant were the most common and also no significant difference in antibody response was observed according to the CSP variant present at the time of infection. Significant correlations between the levels of these interleukins with parasitemia and levels of antibodies against variants of CSP were not observed. In addition, the CSP variants did not influence plasma cytokine levels and there were no positive associations between IL1 and IL2 SNPs and their plasma levels. The results may contribute to the identification and effective participation of human genes in the modulation of the immune response, essential in the establishment of immunization strategies against the disease, in an active transmission area in the State of Pará.

Differential Display technique was used for gene profiling in trnasformed human umbilical vein endothelial cell line (ECV 304) and primary human umbilical vein endothelial cells (HUVECs) to study the cellular response to viral infection. After screening the mRNA from uninfected and infected HUVECs and ECV 304 cells with 16 different random primers we identified 8 gene targets. These genes included the human inhibitor of apoptosis-1 (h-IAP1), 2'-5' oligoadenylate synthetase (2'-5' OAS), 2'-5' oligoadenylate synthetase-like (2'-5' OAS-like), Galectin-9 (Gal-9), MxA, Mx1, Regulator of g-protein signaling (RGS2) and endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN). We found that HUVECs were a better model to study gene expression dureing dengue 2 virus infection but not the transformed cell line, ECV 304. Of the 41 primer combinations utilized in ECV 304 cells detected only one upregulated gene, h-IAP1 and 8 out of the 16 primer combinations tried for HUVECs. We hypothesize the activation of two novel signaling pathways (Tumor necrosis factor- alpha (TNF-alpha), Interleukin1-beta (IL1-beta) in endothelial cells during D2V infection. ALso, our data detected genes that are activated in the Type-I IFN (IFN alpha/beta) signaling pathway during dengue 2 virus infection in HUVEC.

Prostate cancer remains a significant health concern for men throughout the world. Accumulating epidemiologic and molecular evidence suggests that inflammation is an important component in the aetiology of prostate cancer. Supporting this hypothesis, population studies have found an increased risk of prostate cancer in men with a prior history of certain sexually transmitted infections or prostatitis. More general evidence of a relationship between inflammation and prostate cancer has been provided by reports indicating that daily use of non steroidal anti-inflammatory drugs (NSAIDs) may be associated with a lower incidence of prostate cancer. The exact mechanism whereby inflammation might act in tumour development and progression remains to be elucidated, but is likely to be complex. The genetic contribution to inflammatory responses involved in the development of prostate cancer has not yet been extensively or systematically studied. However, this thesis evaluates the role of various inflammation-related genes in the pathogenesis of prostate cancer. The macrophage scavenger receptor 1 (MSR1) is a transmembrane protein that is mainly expressed by macrophages. This receptor mediates the binding, internalization and processing of a wide range of macromolecules, and is suggested to play a major role in the recognition and clearance of pathogenic and damaged cells. Recent reports have suggested MSR1 to be a candidate gene for hereditary prostate cancer. Therefore, we screened the MSR1 gene among men with hereditary prostate cancer and identified 18 sequence variants. One previously reported truncation mutation was found more frequently in men with prostate cancer than in unaffected men, in accordance with previously published results. However, the difference in frequencies we found between these groups was not statistically significant. In addition, we genotyped five common polymorphisms in MSR1 in 215 men with unselected prostate cancer and 425 controls. No association between any of the five common variants and prostate cancer were found. We then performed a comprehensive genetic study using extensive populationbased case-control material to evaluate possible associations between sequence variants in inflammation-related genes and prostate cancer. The first gene to be examined was interleukin-1 receptor antagonist (IL-1-RN), encoding a cytokine that plays an important role in regulation of the inflammatory response by binding to the IL-1 receptor and thus inhibiting the binding of the pro-inflammatory cytokines IL-1α and IL-1β. Collectively, these three cytokines exert a central role in the protection against diverse lesions, ranging from microbial colonisation to infection and malignant transformation. The genetic analysis of IL-1RN revealed that the most common haplotype was significantly associated with prostate cancer risk for patients with prostate cancer, and further this association appears to be stronger in cases with advanced disease. The macrophage inhibitory cytokine-1 (MIC-1), a member of the transforming growth-factor-β superfamily has been shown to exert diverse biological functions, including regulation of macrophage activity in the inflammatory response and both growth inhibition and induction of apoptosis in epithelial and other tumour cell lines. The genetic analysis of MIC-1 revealed that a seuqence variant (H6D) appears to be associated with a decreased prostate cancer risk. We also performed measurements of MIC-1 serum levels among patients with prostate cancer and healthy controls. These data indicate that serum MIC-1 levels are associated with an increased risk for prostate cancer. Further, the clear relation between clinical stage and MIC-1 level also suggest that MIC-1 may be useful as a prognostic factor, where high serum concentration is associated with a poor prognosis. In summary, our results provide further support for the assumption that polymorphisms in inflammatory genes play critical roles in prostate cancer susceptibility. Additional studies are needed to elucidate the mechanisms whereby the demonstrated variations contribute to prostate cancer development.